This invention relates to an improved method for performing the sequential degradation of peptide chains for the purposes of analyzing the constituents of the chains and, more particularly, is directed to the added automated capability of the conversion reaction of the Edman chemistry.
It is important to analyze the amino acid sequence of proteins and peptides in order to understand their biological functions. For example, the function of insulin is dependent upon its particular amino acid sequence. A variety of techniques may be used to determine the linear order of amino acids. One of the most important sequential methods now being used is called the Edman process. The Edman process was originally described in Acta Chem. Scand. 4, 283 (1950). Four later articles describe a general form of its use: Blomback et al., "Human Fibrinopeptides Isolation, Characterization, and Structure," Biochem, Biophys. Acta, 115 (1966) 371-396; Edman and Begg, "A Protein Sequenator," European J. Biochem. 1 (1967) 80-91; Niall et al., "The Amino Acid Sequence of Porcine Thyrocalciton", Proc. of the National Academy of Sciences, Vol. 59 No. 4, pp. 1,321-1,328, April 1968; and Niall, "Sequential Analysis of Proteins and Peptides" Fractions No. 2, pp. 1-10 (1969). Briefly, as discussed in the last article and as shown inside the cover of the Fractions publication, the Edman sequential degradation processes involve three stages: coupling, cleavage, and conversion. In the coupling stage phenyl isothiocyanate (PITC) reacts with the N-terminal amino group of the peptide to form the phenylthiocarbamyl (PTC) derivative. The pH is normally maintained at between 9 and 10 and preferably between 9 and 9.5 for the coupling reaction. In the cleavage step anhydrous acid is used to cleave the PTC derivative, i.e., the anilinothiazolinone (ATZ). After extraction of the thiazolinone the residual peptide is ready for the next cycle of coupling and cleavage reactions. Aqueous acid is used to convert the thiazolinone to the phenylthiohydantoin (PTH) which may be analyzed in an appropriate manner such as by chromatography.
At the end of the coupling step the excess PITC and the organic constituents of the coupling buffer, which is used to maintain the desired environment (such as pH) for the coupling reaction, are removed by extraction with benzene. Certain breakdown products of PITC such as aniline and phenylthiourea are also removed. Diphenylthiourea, another side product, is poorly soluble in benzene but may be removed by further extraction with a more polar organic solvent such as ethyl or butyl acetate. The water from the coupling buffer must also be removed, for example by lyophilization. The reaction vessel is then subjected to a vacuum purging operation to remove any remaining undesired materials and the dried protein as its PTC derivative is then ready for cleavage.
During the cleavage operation the protein is dissolved in anhydrous acid, for example trifluoroacetic acid. Thus, there is a strong shift in pH from basic for the coupling reaction to acidic for the cleavage reaction. The acid is then evaporated and the cleaved thiazolinone derivative is extracted from the residual peptide with butyl chloride or ethylene dichloride. After evaporation of residual solvent and a further vacuum purging operation, the peptide or protein, now one residue shorter, is ready for the next cycle.
In the typical apparatus used to perform the coupling and cleavage steps, each series constituting a cycle, the steps may be broken down into seven stages. A typical apparatus for accomplishing this is the Beckman Protein Peptide Sequencer. The first of the seven stages is the coupling of the protein. This is followed by two wash stages giving precipitation of the sample along with a primary extraction of excess reagents. The cleavage stage, cleaving the amino acid residue on the end of the protein comes next. This is followed by a first extraction which extracts the cleaved amino acid residue. If desired, to be sure that all of the amino acid residue has been cleaved, a second cleavage step may be used followed by a second extraction step.
Reference is made to U.S. Pat. No. 3,725,010 which describes in detail the above-referenced Beckman Protein Peptide Sequencer. This particular type of automatic protein/peptide sequenator avoided or minimized many of the difficulties and limitations experienced by the early efforts to automate a process for determining the amino acid sequence in protein. Several drawbacks were evident in the Edman protein sequenator. The Beckman sequencer includes a reaction cell or chamber having a rotating or spinning cup driven with the chamber. The chamber has to be properly insulated and sealed in order to ensure proper operation. In fluid communication with the reaction chamber are means for introducing and removing gases and liquids. Insulation of the chamber is necessary in order to provide a constant, uniform temperature critical for the reaction. Also, it is necessary to properly seal the reaction chamber so that during the evacuation step for waste removal and for drying, the proper vacuum environment is maintained within the chamber without having any degrading influence from the surrounding atmosphere. Consequently, it is necessary to specifically design a properly sealed driving mechanism for the spinning cup to ensure the vacuum environment within the reaction chamber. One approach as shown in the referenced patent is a magnetic coupling drive unit which is properly sealed. Included within the system are numerous vacuum seals that are necessary in order to ensure the proper environment when the vacuum is necessary. Also, rather high quality and precise vacuum pumps are necessary in order to provide the requisite vacuum when necessary. Also included are numerous vacuum lines and valves. All of this equipment to support the vacuum necessary in the reaction chamber requires a significant maintenance over a period of time.
Reference is made to copending patent application Ser. No. 458,226 filed on Jan. 14, 1983, entitled "An Improved Apparatus for Sequencing Peptides and Proteins", having the same inventors as the present application and assigned to the same assignee as the present invention. In this referenced copending patent application, discussion is directed to an improved apparatus which does not require a vacuum for operating the system. This reduces the maintenance requirements with respect to having numerous vacuum lines, valves and vacuum pumps.
When using the Edman chemistry with systems discussed and referenced in the above copending patent application, one area of the chemistry which is typically not done automatically, but is done manually, is the chemical conversion of the labile anilinothiazolinone derivative of the sequenced amino acid to a more stable phenylthiohydantoin form. This manual conversion process is done outside of the instrument by a technician.
Some existing instruments have incorporated a reaction chamber in which the conversion process occurs. Reference is made to U.S. Pat. No. 3,959,307 which utilizes an intermediate reaction vessel. A complex glass blown vessel is used as the conversion reaction vessel and is wrapped in a water bath. This glass blown reaction vessel with a water bath sheath requires considerable glass blowing artwork. This particular type of device only allows the thermostatting of the conversion process at a single temperature. In addition, the placement of an intermediate reaction vessel between the reaction chamber and the fraction collector results in fluid flow resistance added to the connectors during liquid transfers because of the low differential pressure utilized in the instrument. Further, this intermediate reaction vessel being common for each cycle would tend to contribute to cross contamination between successive cycles of the reaction process unless the vessel is thoroughly washed at the completion of each cycle.
Another system which attempts to somewhat automate the conversion process is the Sequamat P-6 Autoconverter which is discussed in the Sequemat P6 Auto Converter Instruction Manual, Jan. 1, 1978. This particular system uses an intermediate reaction vessel between the spinning cup of the reaction chamber and the fraction collector. This requires added length of the fluid transfer system and more fittings which will create a flow resistance during the transfer of the anilinothiazolinone derivative from the sequencer spinning cup. By using an intermediate reaction vessel it is necessary to provide solvent washing after each conversion so that there is no cross-contamination of the fractions.
It should also be noted with respect to the above-described existing methods for automating the conversion step a control of temperature that must be accomplished by means of circulating water or air systems. As a result, the reaction time for temperature change during the conversion process is limited. The utilization of different temperature levels is somewhat restricted because of the limited temperature change response with a circulating water or air system. These systems require additional fittings and added delivery tubing incorporated into a reaction vessel.